There is an increasing demand to be able to identify and quantify components of mixtures. The greater the complexity of the mixture, the greater the interest in being able to simultaneously detect a plurality of the components present. As illustrative of this situation is DNA sequencing, where it is desirable to efficiently excite from one to four fluorescently tagged components with a laser source, while providing for fluorescent signal emission at a plurality of distinctive wavelengths, where the fluorescence signals should be as intense as possible. In this situation, the different labels should not adversely affect the electrophoretic mobility of the sequences to which they are attached.
Currently, there are four methods used for automated DNA sequencing: (1) the DNA fragments are labeled with one fluorophore and then the fragments run in adjacent sequencing lanes (Ansorge et al., Nucleic Acids Res. 15, 4593–4602 (1987); (2) the DNA fragments are labeled with four different fluorophores and all the fragments are electrophoretically separated and detected in a single lane (Smith et al., Nature 321, 674–679 (1986); (3) each of the dideoxynucleosides in the termination reaction is labeled with a different fluorophore and the four sets of fragments are run in the same lane (Prober et al., Science 238, 336–341 (1987); or (4) the sets of DNA fragments are labeled with two different fluorophores and the DNA sequences coded with the dye ratios (Huang et al., Anal. Chem. 64, 2149–2154 (1992).
All of these techniques have significant deficiencies. Method 1 has the potential problems of lane-to-lane variations in mobility, as well as a low throughput. Methods 2 and 3 require that the four dyes be well excited by one laser source and that they have distinctly different emission spectra. In practice, it is very difficult to find two or more dyes that can be efficiently excited with a single laser and that emit well separated and intense fluorescent signals.
As one selects dyes with distinctive red-shifted emission spectra, their absorption maxima will also move to the red and all the dyes can no longer be efficiently excited by the same laser source. Thus, the detection sensitivity for these dyes will suffer. Also, as more different dyes are selected, it becomes more difficult to select all the dyes such that they cause the same mobility shift of the labeled molecules.
It is therefore of continued interest that improved labels be developed which have strong absorption at a common wavelength, have a high quantum yield for fluorescence, have intense fluorescence signals and have a large Stokes shift of the emission.
Relevant Literature
U.S. Pat. No. 4,996,143 reports the preparation of oligonucleotide probes comprising donor and acceptor fluorophores designed for the detection of complementary DNA target sequences by hybridization to form labeled double-strand DNA fragments. These probes were specifically labeled in the middle of the probe, explicitly excluding the 5′ or 3′ end base unit.
Smith et al., Nucleic Acids Research (1986) 321:674–679 reports the synthesis of oligonucleotides having an aliphatic amino group at the 5′ terminus, as well as the preparation of fluorescent derivatives thereof, containing only a single fluorescent label.